1. Field
The present invention relates to protein sequences encoding antigenized swine antibodies for Foot-and-Mouth Disease (FMD), Foot-and-Mouth Disease Virus (FMDV), or their epitopes.
2. Background
Antibodies are proteins produced by the body's immune system in response to foreign elements known as antigens, which invade the body. Antibodies can elicit an immune response, which subsequently protects the host from infection by the disease-causing agents (antigens).
Foot-and-Mouth Disease, also known as FMD hereinafter, is a highly contagious, severely debilitating disease that infects all cloven-hoofed animals. It is endemic in many developing countries worldwide. In particular, swine in Asia have often been affected by FMD. FMD reduces livestock, productivity, incurs high vaccination costs, and restricts the international trade of livestock and livestock products. FMD is a viral infectious disease and the foot-and-mouth disease virus, also known as FMDV hereinafter, is a small animal virus having a single stranded positive sense RNA genome of about 8,000 nucleotides.
Two major types of vaccines have been produced against FMD. They are conventional vaccines and synthetic peptide vaccines. Conventional vaccines against FMDV use either inactivated FMD virus or a live attenuated FMD virus. The conventional vaccine approach, although generally effective, have several undesirable drawbacks associated with it.
Firstly, it is cost inefficient. This type of vaccine is made from large amounts of live, infectious virus. Maintaining and processing a large quantity of infectious virus is expensive, labor intensive and space inefficient.
Second and most importantly, these vaccines are potentially dangerous. Most of the outbreaks of FMD in recent years have been caused either by the escape of virus from vaccine production units or the use of incompletely inactivated or insufficiently attenuated virus. For example, during the 1980's, a number of outbreaks occurred in European countries including Italy, United Kingdom, France and in Taiwan in 1996. As the causative viruses of outbreaks were often found to be closely related or even identical to the strains that were used in Europe for manufacturing, it is possible that the primary outbreaks were caused by inadequately inactivated vaccines or by virus that escaped from vaccine production plants.
Another problem associated with producing conventional vaccine is that they are thermolabile. Conventional FMD vaccines are relatively unstable when exposed to elevated temperatures and they have to be stored at low temperatures. Constantly maintaining the required low temperatures is often not easily achievable, especially in tropical countries.
An addition potential problem in a virus culturing procedure of the conventional vaccine production vaccine production process is the use of fetal bovine serum virus culturing. It is possible that diseases can be introduced from the fetal bovine serum and affect the vaccinated animals.
Yet another major disadvantage of using conventional FMD vaccines is that most vaccines produced using the conventional method are relatively crude preparations of inactivated tissue culture grown virus. This tissue cultural mix may cause serious side effects such as allergic responses and abortions in susceptible stock.
There are newer forms of FMDV vaccines that do not use inactivated virus. They are synthetic peptide vaccines and recombinant protein vaccines. The identification of the immuno-dominant sites on viral protein 1 (VP1) of FMDV provided new ideas for designing synthetic peptide and recombinant protein FMD vaccines. Compared to conventional vaccines, these two types of vaccines are both safe in production and application. They are also very easy to handle, store, transport and can be designed to meet specific requirements.
The study of synthetic FMDV peptide vaccine was started by polymerizing the 141 a.a.-160a.a. peptide from VP1 with either glutaraldehyde or air-oxidized after a cysteine residue was added at each terminus. It was found that uncoupled peptides could be made immunogenic. In 1987, Francis and his colleagues reported that the presence of C-terminal cysteines with a free thiol group largely enhanced the immunogenicity of free 141-160 a.a. peptide. Similar results were also obtained when multiple cysteine residues were added. It was suggested that the presence of a free thiol cysteine residue would allow the formation of peptide dimers leading to a more ordered secondary structure causing immune complex formation in vivo (Francis, 1995). According to this idea, immunogenicity of tandem repeats (Cys 137-162(x2)) was compared to that of a single copy of Cys 137-163 peptide. It was found that tandem repeats of the FMDV peptide were generally more immunogenic than the single copy of disulphide dimers. The addition of a cysteine residue could result in the formation of disulphide tetramer structures, which improved the immune response further.
The concept of multiple copies synthetic peptides was further tested by using Tam's multiple antigenic peptide (MAP) system (Tam 1988). This system allows solid phase synthesis of a peptide antigen onto a branching lysine backbone to produce several polylysine octamer constructs. This system where there are multiple copies of the peptide resulted in greatly enhanced response.
In order to apply multiple copies of the FMDV peptide, recombinant DNA technology has been applied by fusing small peptide sequences to the gene coding for larger proteins. These larger proteins of recombinant vaccine have a number of characteristics. The goal of linking the peptide to the carrier is to provide a completely uniform and defined structure for the presentation of the immunogens as compared with those prepared by chemical cross-linking (Francis, 1991). This approach was first investigated by fusing single or multiple copies of the FMDV immunogenic peptides to the N-terminus of a bacterial protein, beta-galactosidase (Broekhuijsen et al., 1986; Winther et al., 1986). Beta-galatosidase was chosen because it has been shown that antibodies can be elicited against the epitopes from VP1 that are located at the N-terminus, and it also contains several T cell epitopes (Krzych et al., 1982; Manca et al., 1985). The immunogenicity of this multiple copy FMDV peptide-beta-galactosidase recombinant protein is found to be similar to that obtained from using the lysine background system (Broekhuijen et al., 1987).
Multiple peptide presentation was then further developed using FMDV peptide sequence fused to the N-terminus of the hepatitis B virus core antigen (HBcAg) to produce HBc fusion particles. It was reported that this 27 nm hybrid protein particle was able to give full protection to guinea pigs with results that were close to that elicited by inactivated FMDV VP1 142 a.a.-160 a.a. peptide and could protect animals against challenge infections.
Although initially promising, the synthetic peptide approach and recombinant protein vaccine approach appear to have shortcomings. Among these are poor predictability of the tertiary structure and weak immunogenicity. Peptides in solution exist in conformations that may not be always optimal for receptor binding (B-cell receptor and possibly T-cell receptor and major histocompatibility gene products) if specific conformation at the three-dimensional level is required for it to exhibit its intended functions.
In the case in which synthetic peptides that are relatively small in size, they tend to be easily degraded in the body after injection. Therefore, they may not be very effective in providing long term immune response probably because the recombinant protein vaccine fails to exhibit a proper conformation. Also, peptide synthesis is expensive which may lead to high production cost of the vaccine. As mentioned, presentation of the FMDV epitopes on peptide vaccines can be achieved by fusing them to the N-terminus of microbial proteins like beta-galactosidase or HBcAg. However, using beta-galactosidase may elicit a lot of additional and undesirable immune responses (Bona et al., 1994). After repetitive immunization of this recombinant protein vaccine, side effects may occur such as immediate hypersensitivity that can cause severe hay fever and asthma in the animal.
Nucleic acid vaccines or DNA vaccine represent a new approach to the control of infectious agents. These novel vaccines are easier to design and manufacture. Recombinant DNA technology is used to clone DNA sequences encoding the protein or proteins to be used as immunogens into an eukaryotic expression vector.
Antigenized antibodies are antibodies, which are genetically engineered in their variable domains to express epitopes of different antigens. Antigenized antibodies can be used as immunogens that focus the immune response on specific B- or T-cell epitopes. As such, antigenized antibodies can be used as an alternative approach to conventional or synthetic peptide vaccination.
Therefore, an effective kind of vaccine that offers both the possibilities of safety and efficiency is the antigenized antibody vaccine. The process of antibody antigenization consists of grafting peptide epitopes derived from antigens other than immunoglobulins into complementarity determining region (“CDR”) loops of an antibody molecule. Because the CDR loops are exposed at the surface of the antibody molecule, they provide the major contribution to antibody antigenicity. Unlike the synthetic vaccines described above, antigenized antibodies target antigen-presenting cells via the Fc receptor, thereby maximizing antigen presentation by class II major hisocompatibility (MHC) molecules. Also, antigenized antibodies provide B-cells with a continuous source of antigenic peptides for presentation in class I MHC molecules. In addition to immunogenicity at the B-cell level, antigenized antibodies act as processed peptide products to generate Th-cell immunogenicity.